Molecular Evolution and Nucleotide Sequences of the Maize … · 2003. 8. 1. · Molecular...

Copyright 0 1987 by the Genetics Society of America

Molecular Evolution and Nucleotide Sequences of the Maize Plastid Genes for the cy Subunit of CFI (atpA) and the Proteolipid Subunit of CFo (atpH)

Steven R. Rodermel and Lawrence Bogorad

The Biological Laboratories, Harvard University, Cambridge, Massachusetts 021 38 Manuscript received December 8, 1986

Accepted February 16, 1987

ABSTRACT The nucleotide sequences of the maize plastid genes for the a subunit of CFI (atpA) and the

proteolipid subunit of CFo (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:l and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastidencoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the t subunit of CFI) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5 ‘ - and 3‘- transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of “species-specific” regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.

HE genes for the constituent proteins of the T plastid coupling factor for photophosphoryla- tion (CFt-CFo complex) are dispersed between the genomes of the plastid and nucleus. In all species examined to date, atpA, atpB, atpE, atpF, atpH and atpZ (for the a, /3 and E subunits of CFI and subunits I, I11 and IV of CFo, respectively) are encoded in the plastid DNA, while atPC, atpD and atpG (for the y and 6 subunits of CF, and subunit I1 of CFo, respectively) are encoded in the nuclear genome and trans- ported into the plastid post-translationally (e.g., MEN- DIOLA-MORGENTHALER, MORGENTHALER and PRICE 1976; BOUTHYETTE and JAGENDORF 1978; NELSON, NELSON and SCHATZ 1980; NECHUSHTAI et al. 198 1 ; WE~THOFF et al. 198 1 , 1985; WATANABE and PRICE 1982). The plastid-encoded genes appear to be highly conserved in their sequence arrangement among most higher plant plastid DNAs. at@ and atpE are clustered in one transcription unit, either partially overlapping or immediately adjacent to one another, while atpZ, atpH, atpF and atpA are clustered (in this polar- ity) in another transcription unit some distance away from the atpBE locus on the plastid chromosome (e.g., WESTHOFF et al. 1981, 1985; KREBBERS et al. 1982; HOWE et al. 1982, 1983; DEHEJJ et al. 1983; FLUHR,

The sequence data presented in this article have been submitted to the EMBL/GenBank Data Libraries under the accession number Y00310.

Genetics 116: 127-139 (May, 1987)

FROMM and EDELMAN 1983; DENO, SHINOZAKI and SUGIURA 1983, 1984; BOVENBERG et al. 1984; HUTTLY and GRAY 1984; KO, STRAUS and WILLIAMS 1984; ZURAWSKI and CLEGG 1984; OLIVER 1984; PHILLIPS 1985; BIRD et a l . 1985; RODERMEL and Bo- GORAD 1985, 1986). Among higher plants, the nucleotide sequences of all the plastid-encoded coupling factor genes have been reported in tobacco (SHINO- ZAKI et al. 1983a; DENO, SHINOZAKI and SUGIURA 1983,1984) and wheat (HOWE et al. 1982,1985; BIRD et al. 1985; J. C. GRAY, unpublished data). In addition, among angiosperms the sequence of atpBE has been reported in maize (KREBBERS et al. 1982, spinach (ZURAWSKI, BOTTOMLEY and WHITFELD 1982), barley (ZURAWSKI and CLEGG 1984), and pea (WHITFELD, ZURAWSKI and BOTTOMLEY 1983), and the sequences of atpH and atpZ have been reported for spinach (ALT et a l . 1983) and pea (COZENS et aE. 1986), respectively. The nucleotide sequences of atpA and atpH from maize are presented in the current studies.

Although the striking conservation of sequence content and arrangement of the plastid-encoded coupling factor genes is in accord with the notion that the molecular evolution of angiosperm plastid DNAs is distinctly conservative in nature (reviewed in PALMER 1985a,b), little is known about the mode and tempo of sequence change in various regions of the plastid

128 S. R. Rodermel and L. Bogorad

chromosome. Comparative DNA hybridization and restriction site polymorphism analyses have shown that sequences within the large inverted repeat structure (characteristic of most higher plant plastid DNAs) are evolving more slowly than sequences within the single copy regions of the chromosome (PALMER 1985a). However, other than these global studies, detailed investigations of nucleotide divergence in the plastid DNA are extremely limited, and have been restricted in the case of protein-coding genes to comparative studies of rbcL (the gene for the large subunit of ribulose bisphosphate carboxylase), at#, and atpE (SHINOZAKI et al. 1983b; ZURAWSKI, CLEGG and BROWN 1984; ZURAWSKI and CLEGC 1984). These analyses have demonstrated that the patterns and levels of nucleotide substitution are the same in these three genes in comparisons between maize and barley, indicating that each codon position is evolving at about the same rate in these genes in these two monocots (ZURAWSKI and CLEGG 1984). One purpose of this study was to ascertain whether other plastid coupling factor genes are evolving like atpB and atpE in the monocots, and to investigate whether the mode and tempo of sequence evolution of these genes is similar in the dicot, as well as monocot, angiosperm lineages. The data show that the coupling factor genes differ in their patterns of nucleotide substitution both within and between the two lineages, and that these differences are manifested in differing constraints on nonsynonymous or synonymous codon selection.

Another purpose of this investigation was to examine the patterns of nucleotide substitution in the 5 ‘ - and 3 ’-transcribed regions flanking the protein coding sequences of atPA and atpH. Previous comparative analyses of the flanking regions of plastid-coding genes, though limited, have shown that the 5’-flanking region of rbcL is evolving at about half the synonymous substitution rate of the gene itself in comparisons between maize and barley, indicating this region is selectively constrained in its sequence evolution with respect to the third codon position (ZURAWSKI, CLECC and BROWN 1984). The constraints in this region were attributed to conserved sequence requirements for promoter-binding. Northern hybridization experiments have established that atpA and atpH from maize are complementary to several of the same polycistronic transcripts, as well as to numerous other smaller mRNAs, and that the ends of some of these transcripts likely reside in the flanking regions of these genes (RODERMEL and BOGORAD 1985). It was further observed in these studies that all of the transcripts complementary to this region of the chromosome increase in abundance following the illumination of dark- grown seedlings, but that the increases are not uni- form from transcript to transcript. The data thus suggested that unless an extremely complex set of

RNAses change in activity during greening, the flanking regions of atpA and atpH likely contain transcription initiation and/or termination sequences involved in the photoregulation of transcript levels. Since complex transcript patterns have also been observed for the region of the plastid chromosome bearing atpA and atpH in wheat (BIRD et al. 1985) and tobacco (FLUHR, FROMM and EDELMAN 1983; DENO, SHINO- ZAKI and SUGIURA 1984), it was hoped that comparative sequence analyses of these regions might reveal conserved sequence elements that may be involved in transcriptional and/or processing activities. The data in this report indicate, however, that these, and other, putative regulatory elements in these regions are not particularly conserved among these three species. This is reflected in rates of nucleotide divergence in these regions which equal, or vastly exceed, synonymous substitution rates (depending upon the sequences compared). However, the data are not inconsistent with the notion that these regions contain variable numbers of “species-specific” regulatory elements.

MATERIALS AND METHODS

The plasmids pZmc527, pZmc415 and pZrl2 contain Zea mays plastid DNA restriction fragments BamHI 3, BamHI 24, and EcaRI b, respectively, cloned in pBR322 (RODERMEL and BOGORAD 1985). The nucleotide sequences of portions of these fragments containing the genes for the a subunit of CF, (atpA) and subunit 111 of CF, (atpH) (RODERMEL and BOGORAD, 1985, 1986), were determined by the MAXAM- GILBERT chemical cleavage method (1 980).

High resolution S-1 nuclease mapping experiments were performed by procedures modified from BERK and SHARP (1977). Total cell RNA ( 1 50 fig) from 16-hr-greened maize seedlings (RODERMEL and BOCORAD 1985) was mixed with approximately 100 ng of DNA (5’4abeled at a single end) in a total volume of 50 pl hybridization buffer [SOW deion- ized formamide/l mM Na2EDTA (pH 8.0)/0.4 M NaCl/4O mM PIPES (pH 6.4)) The sample was incubated for 10 min at 80” to denature the DNA, and then transferred to 41” for 16 hr. Following the addition of 400 pl ice cold S-1 nuclease buffer [0.28 M NaC1/0.05 M sodium acetate (pH 4.6)/4.5 mM ZnS04/20 pg carrier tRNA], the sample was incubated with S-1 nuclease (75 units, New England Nu- clear) for 30 min at 37”. After phenol/chloroform extrac- tion, the ethanol precipitate was vacuum desiccated, resus- pended in sequencing dye, and electrophoresed through 8% polyacrylamide/7 M urea sequencing gels next to DNA sequencing reactions of control fragments not treated with S- 1 nuclease.

RESULTS

The nucleotide sequences of atpA and atpH The maize plastid genes for the (Y subunit of CF,

(atpA) and subunit I11 (the proteolipid subunit) of CFo (atpH) were sequenced by the strategy outlined in Figure 1. It had been determined previously that atpA and atpH are closely linked on the maize plastid chromosome (RODERMEL and BOCORAD 1985, 1986). The

Molecular Evolution of atpA,H

atpH atpA pslA2 pslA1 k-- - 4 , A q -

129

A

0 '

8

Alu I Bam H I CfoI Dde I Eco R I Fnu 4HI Hind III Hinf I Hpa II Nde I Rsa I Sal I Sau 961 Taq I

100 300 500 200 400 600 800 io00 1200 i400 1600

FIGURE 1.-Map location of atpA and atpH on the maize plastid chromosome, and the strategy used to sequence these genes. A, An 18- kbp portion of the maize plastid chromosome is illustrated [from BEDBROOK and BOCORAD (1976) and LARRINUA et al. (1983)], showing the locations and relative transcriptional polarities of the photoregulated genes for the a! subunit of CF, (&PA), subunit 111 of CFo (atpH), and the duplicated genes for the P700 chlorophyll a proteins of photosystem I (PslAI and psJA2) (RODERMEL and BOGORAD 1985, 1986; FISH, KUCK and BOGORAD 1985). atpH is contained within EcoRI subfragment "s" of BamHI fragment 3 (8.7 kbp), whereas atpA is partially contained within EcoRI fragment "4" of BamHl 3 and extends into BamHl fragment 24 (1.3 kbp), as well as into EcoRI fragment "b." EcoRI fragments "b," "4" and "s," and BamHI fragment 24 served as the sources of plastid DNA in determining the sequences of these genes. B, The nucleotide sequences of atpA and atpH, including portions of their flanking regions, were determined by the MAXAM-GILBERT procedure (1 980). Sequenced fragments include those indicated by arrows.

nucleotide and derived amino acid sequences of these genes are shown in Figure 2: atpA contains 508 codons and encodes a protein with a M , of 55707, while atpH contains 81 codons and encodes a protein with a M, of 7975. The nucleotide sequences of portions of the flanking regions of these genes are also presented in Figure 2. These include approximately 100 bp of both the 5'- and 3'-flanking regions of atpH, the intergenic region between the 5'-end of atpA and the 3'-end of atpF, the gene for subunit I of CFo (RODERMEL and BOCORAD 1986); and the spacer region between the 3'-end of atpA and the 3'-end of the convergently transcribed tRNAArg (UCU) gene (S. RODERMEL, P. ORLIN and L. BOCORAD, unpublished data).

Comparative molecular evolutionary analyses of the coding regions of atpA and atpH

Multiple substitutions at the same nucleotide site and nonrandom mutation bias are two of the most important factors which can distort estimates of the number of nucleotide substitutions which have OC-

curred in two DNA sequences since their divergence from a common ancestor (KIMURA 1981, 1983). TO minimize the former, it has been suggested that two sequences should be at least 85% homologous (BROWN

et al. 1982). For this reason, comparative analyses of atpA and atpH were restricted to sequences of these genes from angiosperms. These include the plastid atpA genes from maize (this report), wheat (HOWE et al. 1985; BIRD et al. 1985; HOWE 1985) and tobacco (DENO, SHINOZAKI and SUGIURA 1983, 1984; DENO and SUCIURA 1984), and the plastid atpH genes from maize (this report), wheat (HOWE et al. 1982; BIRD et al. 1985), tobacco (DENO, SHINOZAKI and SUCIURA 1984), and spinach (ALT et al. 1983). The nucleotide and derived amino acid sequences of these genes are shown in Figure 2; only those sites which differ from maize are indicated. The comparative analyses of these genes are presented in Table 1, and show that among the various sequence comparisons the nucleotide conservation of atpH ranges from 91 to 98%, while that of atpA ranges from 86 to 96%; the levels of nucleotide and amino acid conservation are similar in each comparison.

The possibility of multiple and revertant changes at the same site cannot be discounted even in closely related sequences, and thus numerous equations have been derived to correct for their occurrence (reviewed in LI, LUO and Wu 1985). Since some of these equa-

TOSACCO : WHEAT: KAIZE:

C C A G C G TGGATGATT - - C - - - GG TAA T ~ TTC-TCC-- A __._...___

TTGTACTTATTAGTTACTTCTAC---------CCGATAGAG~A--GAA--GTT

T: - _ C C CA G GT -. -T w: - G A ACT C H: G G A A G T A A T A A ~ ~ C ~ G T A T C ~ A A C C A ~ ~ ~ G A C A C G ~ C T . - - C A T C

c - S: T: W!

G C A G T C

T G

T

n: ATG AAT CCA CTA ATT GCT GCT GCT TCC GTT ATT GCT GCT GGA TTG GCC GTA GGC N : N N P L I A A A S V I A A G L A V G W: T: S s:

S : T G A T G T: A G G T W: A T N : CTT GCT TCT ATT GGG CCC GGA GTT GCT CAA GGT ACT CCT GCA GGA CAA GCT GTA N : L A S I G P G V G Q G T A A G Q A V W’ T: S:

C A C A T G C A C G C

C I%: GAA GGT ATT GCG AGA CAG CCA GAA GCA GAA GGT AAA ATA ACA GGT ACT TTA TTG N : E G I A R Q P E A E G K I R G T L L U: T: s:

T A T A T

W: A H: CTT AGT CTA GCT TIT ATG GAA GCT TTA ACA ATT TAT GGA CTG GTT GTG GCA CTA N : L S L A F N E A L T I Y G L V V A L W: T: S:

5 : T: W: N:

T GT-..--r...-..--------..- A T T GCTT T AAAA-----

CGG CTT TTA TIT GCG AAC CCT TIT GTT TAA TCCTAAAAAAGAAA-ATGACTCCTTTAG T A T-TCTTTCGA C

n : n L L F A N P F V * W: T: S:

T: I T TCA T T A C w: A A A C N: CCT AAA CCT ATT GAT CGG AGA GGC GAA ATT GTC GCT TGA CA4 TCT CGC TTA ATT N : A K P I D G R G E I V A S E S R L I W: Y 1 T: S F

T: G C c T G C C G T w: A A C C T n: CAA TCT CCT GCT CCG GGT ATA ATT TCT AGG CGT TCC GTA TAT GAA ccc CTT CAA n : ~ s P A P G I I s R R s V Y E P L Q w: S 1: A

T: C T T A T A W: T n: ACA GGG CTT ATT GCT ATC GAT TCG ATG ATC ccc ATA GGG CGC GGT CAC CGA GAG ~ : T G L I A I D S N I P I G R G Q R E w: T:

T: C T G C W: n: TTA ATT ATT GGG GAC AGA CAG ACT GCC AAA ACA CCA GTA GCC ACA GAT ACA ATT n : ~ I I G D R Q T G K T A V A T D T I W ’ T:

C A T G A T: W: G G M: CTC AAT CAA AAA GGT CAA GAT GTA ATA TGT GTT TAT GTA GCT ATC GGT CAA AGA N : L N 9 K G Q D V I C Y Y V A I G Q R W: G T: Q N K

1: T T C G C A G AG A G w: A T O n: GCA TCC TCC GTG GCT CAA GTA GTA ACT ACT TTC CAC GAA GAG GGG GCC ATG GAA

T: L Q R

N : A S S V A Q V V T T F H E E G A M E U:

T: G C C A C T W: T

C T C

n: TAC ACT ATT GTA GTA GCT GAA A% GCG GAT TCA ccc GCT ACA TTA CAR TAT CTC M : Y T I V V A E M h D S P A T L Q Y L W: T: 1‘‘

T: A T A T I A C U: n: GCT CCT TAT ACC GGA GCA GCC CTG GCT GAG TAT TTT ATG TAC CGC GAA CGG GAT n : ~ P Y T G A A L A E Y F ~ Y R E R H W: T:

-69

-1

54

108

162

216

273

A

390

444

498

552

606

660

714

768

C

I : - - A A A A T G C C A C G T G T CTT TC ATC G - C W: A T CCC C T - n: A T T A G A T A C ~ ~ A G T A C A T T G G C A ~ ~ T T A ~ A A ~ A A G T - A T A T C A A T A ~ A

T: CTCC CC A T _ _ __._._ T T G - ..______... ......._ A A CC w: m A A C

8: C----TpTACTGCTATTTA ........ T T C A A m A T T A G ~ ~ G T T - C T A ~ ~ A G A A m

1: T ___._ T T T --.- G A C T A W: C G G G---TC N: A G G C A T T A T T T T T - C C C T T G C T T C T T A A A G A M C A C T A ATG GCA ACC Cn: ti:

-+ ~ A T L =

W: T: V I

T: CT G T A T A W: C G T M: CGA GTC GAC GAA A T T AAT AAA ATT CTC CGC GAA CGT ATT GAA CAA TAT AA1 AM:

U: H V L T: A S N I

n : R v D E I N K I L R E R I E Q Y N R

T:G AA TA C AC C T A G C C W: A T N: AAA GTA GGG ATT GAG AAT ATC GGT CGC GTA GTG CAA GTG GGG GAT GGG ATT GCT n : K v G I E N I G R V V Q v G D G I A W: T : E K V T T L

T: GAG A 0 G G A G W: n: CGT ATT ATA GGT CTT GGT GAA ATA ATG TCA GGT GAA TTA GTC GAA m GCA CAA n : ~ I I G L G E I n s G E L V E F A E w: T: H D V A E

T : T A T C A T T G W : T T G C N: GGG ACG AGA GCT ATT GCT CTG AAT TTG GAA TCT AAA AA1 G ’ n GGC ATT GTA I T A M : G T R G I A L N L E S K N V G I V L W: 1: I N V

T: T A T A C G W: n: ATG GGC GAT GGG TTG ATG ATA CAA GAG GGA ACT m GTA AAA GCA ACA GGA AGA n : n G D G ~n I Q E G s F V K A T G R W: T: L 5

1: w. c c

T I T

C G G G . ..

N: ATT GCT CAG ATA CCC GTG AGC GAG GCT TAC TTG GGT CGT GTI ATA AAT GCT CTC N : I A Q I P V S E A Y L G R V I N A L w: P V T:

1: T C C A G W: T N: ACC TTA ATA ATT TAT GAT GAT CTC TCC A M CAG GCA CAA GCT TAT CGC GAA ATG N : T L I I Y D D L s K Q A Q A Y R Q n W: T: P

T: T C G T T A W: T G C n: TCC CTT CTA TTA AGA AGA CCT ccc GGC CGC GAA GCT TAT CTA GGG GAT GTT m n : s L L L R R P P G R E A Y L G D V F W: P 1:

T: G G AG T W: N: TAT TTG CAT TCA CGC GTT TTA GAA AGA GCC GCT AAA TTA AA1 TCT cT1‘ TTA GGC N : Y L H S R L L E R A A K L N S L L G W: T: s s

T: A c T A C G T G W: A N: GAA GGG AGT ATG ACC GCT TTA CCA ATA GTG GAG ACT CAA TCT GGA GhC GTT TCC

W: T:

T: T C T G W: c c T

T

~ : E G S ~ T A L P I V E T Q S G D V s

n: GCC TAT ATT CCT ACT AAT GTA ATT TCA ATT AWL GAT CGA CAA ATA TTC TTA TCC U : A Y I P T N V I S I T D G Q I F I S W: 1:

I T C A c c T: C C U: C T N: GCG GAT CTA TTC AAT GCC GGA ATE CGA CCT GCT ATT AAT Gn: GGI A T T TCA G‘K M : A D L F N A G I R P A I N V G I S V W: T: s

T: G G A T W: G n: TCC AGA GTA GGA TCC GCA m CAA ATT AAA ccc ATG AM GAA CTA GCT GGC AAA n : s R V G s A A Q I K A ~ K Q V A G K U: T:

T: T A A G T T W: 0 n: TCA AM TTG GAA CTA GCT CAA TTC GCA CM TTA CAA GCC m GCA GAA TIC GCC n : s K L E L A Q F A E L Q A F A Q F A U: T: L E

343

- 5 1

12

66

120

174

228

282

336

B

822

876

930

984

1038

1092

1146

1200

D

FIGURE 2.-The nucleotide and derived amino acid sequences of the noncoding strands of atpA and atpH from maize plastids. Sites which differ from maize in other angiosperm atpA and atpH sequences are indicated above (for nucleotides) or below (for amino acids) the maize sequence (see text for references). The sequences were aligned to maximize homologies; gaps in one sequence versus another are indicated by dashes. Putative Y S ~ ~ ~ ~ - D ~ ~ ~ ~ ~ ~ ~ w ribosome-binding sites are designated by double underlines, and RNA termini identified by S 1 nuclease mapping analyses (Figure 3) are indicated by vertical arrows. Putative “-1 0” and “-35” promoter elements are underlined upstream from these termini.

Molecular Evolution of atpA,H 131

T: T A c G T C A A T

P: TCC GCT CTG CAT AM ACA AGT CAG AAT CAA TTG GCA AGG GGT GGA CGA TI% CGG n : s A L D K T S Q N Q L A R G R R L R w:

w: T c A 1254

T: D A T Q T: GCT A G A A A X U: G G T A n: GAA TTG CTT AM CAA TCC WVL TCA AAC CCI CTC CCA GTC GAA GAG CAG GTA GCT n : E L L K Q s Q s N P L P V E E Q V A U: A I T: A T I U

T: A AC C A C G F : A U: ACT ATT TAT ACC GGA ACG AGA GGA TAT CTT GAT TCG mA GAA ATT GAA CAG GTA n : T I Y T G T R G Y L D s L E I E Q v U: T: N V G

T: G T T c CT T T AC A G w : T T n: AAG AM TTT CTG GAT GAG TTA CGT AM CAC CTA AAA GAT ACT h4A CCT CUL TTC ~ : K K F I D E L R K H L K D T K P Q F w: N T: R V T Y T N

T: C cc T G G U: C G T n: CM GM ATT ATA TCT TCT AGT AAG ACA mc ACC GAG CAA GCA GAA ACC CTT TTG 1470 U : Q E I I S S S K T F T E Q A E T L L U: I T: T E A

T: A A A G C T ATA G w: G C T T ._ n: AAG GAA GCT ATT w GAA CAG cm GAA CGG m TCC CTT CAG GAA CAA ACA TAA 1526 n : K E A I Q E Q L E R F S L Q E Q T * w: * - . * T: H D I A *

T: G A TAT ATC T G CTTA CTT-----AATM A A -.--.- GCG C GG U: T T ..________..__. T GTTGAG

1308

1362

1416

n: A T A T ~ ~ ~ C A T G T C T A C T T C C T G ~ A G T A G A A G A G ~ T C . - - . . - M A G A ~ C A T 1589

T: CA AT . -CAG C C T C----------- C M C AAMMT T G w: MAG C T ITTAG _.__. n: T T G M T C A T G C * M . T C G ~ A G . - - - - T A T ~ A ~ A M . ~ ~ - - G A A T A G A T A G A M 1649

T: T - U: T - n: AMGAT 1655 E

tions presume that mutational processes are random, while others correct for nonrandom mutational biases, the sequences in Figure 2 were systematically compared with one another to determine if either transition (G-A, T-C) or transversion (A-T, C-G, A-C, T-G) mutational biases predominate in the sequence evolution of atpA and atpH from higher plants; if entirely random, transversions should occur twice as frequently as transitions. The data in Table 1 show that transitions occur about twice as frequently as transversions. Consequently, formulas of KIMURA (198 l), which correct for this bias, were used to estimate the average number of nucleotide substitutions per site ( “ K ” ) in each sequence comparison; the average number of substitutions at the first, second and third codon positions (”KI,” “K2” and UK3,n respectively); the average number of silent (synonymous) nucleotide substitutions at the third codon position (“K,”); and the average number of amino acid-altering (nonsynonymous) nucleotide substitutions at the first and second codon positions (“K,”). The proportion of synonymous and nonsynonymous nucleotide substitutions in each sequence comparison was also tabulated directly (“Per- cent silent substitutions” in Table 1).

The data in Table 1 show that the fewest number of nucleotide substitutions occur at the second codon position and the greatest number at the third codon position in each of the nine sequence comparisons (K1 vs. K 2 vs. K3); as expected, most of the changes at the

third position are silent (K, is approximately equal to K s , within the limits of confidence). Contrasting the patterns of nucleotide substitution in atpA vs. atpH, the data indicate that the average number of nucleotide substitutions ( K ) is higher in atpA than atpH in each of the three comparisons involving species in which both genes have been sequenced. Chi square heterogeneity analyses have shown that these differences are statistically significant, although the difference in K between atpA and atpH appears to be much less significant in the maize/wheat comparison due to the relatively small number of nucleotide differences in atpH from these two species (Chi square data not shown). Regardless, the higher nucleotide substitution values in atpA are primarily a consequence of higher levels of nonsynonymous substitution (K,), since synonymous substitutions at the third codon position (K,) are statistically homogeneous for both genes in each of the three species comparisons. It should be pointed out, however, that the relative proportion of nonsynonymous substitutions in atpA versus atpH is actually much higher than that indicated by comparisons of K,, since the K , values are serious overestimates of nonsynonymous substitutions in atpH. This arises from the assumption in calculations of K , that nearly all substitutions at the first codon position are nonsynonymous. In fact, the vast bulk of substitutions at this site in atpH are synonymous changes; only a single amino acid-altering change is observed in all of the atpH comparisons in Figure 2. The data in Table 1 thus show that the patterns of nonsynonymous substitution differ markedly in atpA vs. atpH, and indicate that the second codon positions of these genes are evolving at different rates.

To examine the mode and tempo of sequence evolution of atpA and atpH in greater detail in the monocot and dicot lineages of angiosperms, rates of nucleotide substitution (per l O9 yr) were calculated from the K , K,, and K , values in the maizelwheat and tobacco/spinach comparisons (Table l) , assuming divergence times of 60 X lo6 yr for maize and wheat (STEBBINS 1981), and 60-100 X lo6 yr for spinach and tobacco (MULLER 198 1). Because these rates could not be calculated for dicot atpA genes (since only a single dicot atpA sequence has been reported), rate estimates derived from comparative sequence analyses of atpB and atpE, similar to those reported in Figure 2 and Table 1 , were included in Table 2. It is worth pointing out, however, that the nucleotide substitution rates for atpA are likely similar to those for atpB among the dicots, since the patterns of nucleotide substitution in these two genes are very similar in comparisons between maize, wheat, and tobacco (as in Table 1).

The data in Table 2 reveal that there are no statistically significant differences within the monocots


TABLE 1

Nucleotide divergence in the coding regions of atpA and atpH

Amino acid homology

Gene n (%i

Maize/Wheat atpA 1515 97

atpH 246 100

Maize/Tobacco atpA 1524 88

atpH 246 99

Wheat/Tobacco atpA 1515 87

atpH 246 99

Maize/Spinach atpH 246 100

Wheat/Spinach atpH 246 100

Tobacco/Spinach atpH 246 99

Nu- cleic acid

homol-

:!2J 96

98

86

92

86

91

92

93

92

Transition/ Transversion

38/22 = 1.7

4/1 = 4.0

127/83 = 1.5

13/8 = 1.6

135/81 = 1.7

15/7 = 2.1

13/6 = 2.2

12/5 = 2.4

13/8 = 1.6

Percent silent

substitution

75

100

63

95

60

95

100

100

95

K K1

0.0408 0.0201 (0.0053) (0.0064) 0.0207 0.0123

(0.0093) (0.0124)

0.1539 0.0790 (0.01 11) (0.0130) 0.0912 0.0507

(0.0204) (0.0256)

0.1601 0.0883 (0.01 14) (0.0139) 0.0962 0.0377

(0.021 1 ) (0.0220)

0.0822 0.0643 (0.0197) (0.0295)

0.0731 0.0513 (0.0181) (0.0263)

0.0913 0.0643 (0.0204) (0.0295)

K2

0.0100 (0.0045)

0

0.0726 (0.0125)

0

0.0842 (0.0136)

0

0

0

0

K3 K ,

0.0958 0.0805 (0.0146) (0.0129) 0.0513 0.0513

(0.0263) (0.0263)

0.3465 0.2929 (0.0336) (0.0307) 0.2484 0.2089

(0.0653) (0.0588)

0.3424 0.2969 (0.0339) (0.0135) 0.2869 0.2473

(0.0731) (0.0674)

0.1972 0.1647 (0.0560) (0.0497)

0.1805 0.1480 (0.0527) (0.0461)

0.2293 0.1825 (0.0612) (0.0871)

K.

0.0150

0.0062

0.0758

0.0253

0.0862

0.0188

0.0322

0.0256

0.0322

“n” is the total number of homologous nucleotide positions that were compared in each pair of sequences, excluding gaps in one sequence US. another. The total number of transition and transversion events in each sequence comparison is shown in the form of a ratio, while only the percentage of silent substitutions is shown for each comparison. In determining the latter value, each nucleotide substitution was considered as an independent event in those relatively few cases in which a given codon contained more than one nucleotide change. Estimates of evolutionary distance between the two sequences (in substitutions per nucleotide site) are presented on the top line in each comparison and their error variances (due to sampling errors) are given below in parentheses. K, K 1 , K I and K S were calculated according to equation (1 2), of KIMURA ( 1 98 1). K , and its error variance were calculated according to equations (1 1) and (1 3), respectively, of KIMURA (1981). The average number of nonsynonymous substitutions (K.) was estimated by K, + K2/2 (KIMURA, 1981).

TABLE 2

Rates of nucleotide substitution in plastid coupling factor genes (substitutions/site/lOg year)

Monocot/Monocot atpA atpB atpE atpH

Dicot/Dicot atpA atpB atpE at;bH

Nonsynonymous (K0/2T)

0.13 0.17 0.06 0.05 -

0.1 7-0.28 0.38-0.63 0.16-0.27

Synon mous

0.67 0.86 0.91 0.43

(K,J;T)

- 1 .Ol-1.68 1.14-1.90 0.90-1.50

Total ( K I 2 T )

0.34 0.42 0.38 0.17 -

0.46-0.77

0.46-0.76 0.64-1.07

~ ~~

Amino acid Nucleic acid homology (%) homology (W)

97 96 97 95 98 96

100 98 - - 95 91 87 88 99 92

Rates of nucleotide substitution in plastid coupling factor genes. The rates (X 1 O-’ substitutions/site/year) were calculated by dividing K, K , or K , by 2T (KIMURA, 1981), where T is the estimated divergence time between two species. Rates were calculated at the lower and upper bounds of 7’ in the spinach and tobacco comparisons. The nucleotide substitution values were obtained from Table 1, or from comparative sequence analyses of the plastid atpB and atpE genes from maize (KREBBERS et al. 1982), wheat (HOWE et al. 1985), tobacco (SHINOZAKI et al. 1983a), and spinach (ZURAWSKI, BOTTOMLEY and WHITFELD 1982).

“Dicot atpA comparisons cannot be made.

among the four genes in any of the three rate comparisons, with the exception that the sequence evolution (K/2T and K,/2T)(T = estimated divergence time between species of atpH) may be proceeding at a lower rate than the others (consistent with the data in Table 1). This is reflected in higher levels of nucleotide and amino acid conservation of atpH than are observed

for the other three genes in the maize/wheat comparison. There are also no statistically significant differences within the dicots among atpB, atPE and atpH in any of the three rate comparisons, with the exception that atpE has a higher rate of nonsynonymous substitution (K, /2T). This is reflected in a much lower level of amino acid homology between the E subunit pro-


teins from tobacco and spinach (87%), than between the 0, or proteolipid, subunit proteins from these species (95 and 99%, respectively).

Contrasting the rates of nucleotide substitution in these genes in the monocots vs. the dicots, the data in Table 2 indicate (with a few exceptions) that each of the three rates is about the same in both lineages when a divergence time of 100 million years between spinach and tobacco is used in the rate calculations (e.g., K/2T ranges from 0.34 to 0.46 in both lineages, excluding values for atpH in the monocot comparison and atpE in the dicot comparison). In contrast, each of these rates (with exceptions) is about twofold higher in the dicots, assuming a divergence time of only 60 million years between spinach and tobacco. Although more precise divergence time estimates will be needed to differentiate between these two possibilities, the data nevertheless indicate that the sequence evolution of atpE and atpH has decreased markedly in the mon-

1. The rate of nucleotide substitution (K/2T) in atpE is about two- to threefold higher in the dicots than in the monocots (depending upon the divergence time of spinach and tobacco). Most of this higher rate of sequence evolution can be attributed to a six- to tenfold higher rate of nonsynonymous substitution K,/2T) in the dicots, since the rate of synonymous substitution in atpE is at most twofold higher in the dicots (again, depending upon the divergence time). These differences in K/2T and K,/2T are statistically significant, and are reflected in much higher levels of nucleotide and amino acid sequence conservation of atpE in the monocots than dicots (e.g., in contrast to more similar levels of conservation of atpB in both lineages). An apparent enhancement in the sequence conservation of atpE in the monocot lineage has been noted previously (PALMER 1985a).

2. The rate of nucleotide substitution (K/2T) in atpH is about 2.5-4.5-fold higher in the dicots than in the monocots (again, depending upon the spinach/ tobacco divergence time). Most of this higher rate of sequence evolution can be attributed to at least a two- to threefold higher rate of synonymous substitution in the dicots, since the rates of nonsynonymous substitution are about the same in both lineages. (As stated earlier, the K, values are overestimates of nonsynonymous substitution in the case of atpH). These differences in K/2T and KJ2T between the monocot and dicot atpH sequences are statistically significant, and are reflected in a higher level of nucleotide conservation of this gene in the monocots than in the dicots (98% vs. 92%), in contrast to similar levels of amino acid conservation of the proteolipid subunit in both lineages (100% vs. 99%).

Comparative molecular evolutionary analyses of the flanking regions of atpA and atpH

The nucleotide sequences of portions of the 5’- and 3’-flanking regions of atpA and atpH from maize,

ocots:

wheat and tobacco are illustrated in Figure 2. North- ern hybridization experiments have shown that these regions are complementary to polycistronic and other mRNAs (which, however, vary in number and size from species to species), and that the ends of some of these transcripts likely map in these regions (FLUHR, FROMM and EDELMAN 1983; DENO, SHINOZAKI and SUGIURA 1984; BIRD et al. 1985; RODERMEL and Bo- GORAD 1985). The data in Figure 2 indicate that there are numerous insertions (deletions) in one sequence us. another. These insertions range in size from 1 to 20 bp, though the majority are short (3-5 bp), and most are flanked by perfect (or near-perfect) direct repeats. For example, of the five gaps in the 5’- flanking region of atpH from wheat us. maize, two are single nucleotide insertions in the maize sequence, while two are short insertions in the wheat sequence (“ACT” and “TTCTCC”) and one is a short insertion in the maize sequence (“GAAGTTG”); each of these latter three sequences is repeated immediately upstream and/or downstream in both wheat and maize. Similar events have been observed in the flanking regions of other plastid genes, and it has been suggested that they arise by mispairing or slippage of DNA strands during replication or repair (PALMER 1985b). It might be pointed out, however, that optimal levels of A and T may facilitate the generation of such events, since in the present studies these events predominate in the intergenic spacer regions (which range from 67 to 75% AT) us. the coding regions of atpA and atpH (57% A T for both genes).

T o determine the patterns of nucleotide substitution in the transcribed flanking regions of atpA and atpH, the number of transitions and transversions was tabulated in each sequence comparison (excluding gaps), and the average number of nucleotide substitutions per site (K) was calculated (Table 3). The data in Table 3 show that the transition/transversion ratio approaches random expectations (TJT, = 0.5) in most comparisons. The lack of a pronounced transition bias in these regions (in contrast to the 2:l transition bias in the coding regions of these genes) may be an artifact arising from small sample sizes in some of these comparisons. On the other hand, the lack of such a bias in those comparisons involving a relatively large number of substitution events may be a reflection of the ease with which the record of transition changes (but not transversion changes) can be obscured with evolutionary distance (BROWN et al. 1982).

Due to the presence of numerous gaps, it is difficult to estimate precisely the overall degree of nucleotide divergence in the transcribed flanking regions of atpA and atpH. Nevertheless, several general conclusions can be drawn from the data in Table 3. In the first place, if gaps are included in the calculations (assuming a gap to be equal to one substitution event), the


TABLE 3

Nucleotide divergence in the flanking regions of atpA and atPH

5’ Flanking 3’ Flanking

Gene n Gaps Ts/Tu K n Gaps Ts/Tu K

Maize/Wheat atpA 89 2 6/3 = 2.0 0.1098 122 6 2/7 = 0.3 0.0779 (0.0377) (0.0264)

atpH 110 5 2/3 = 0.7 0.0469 112 1 9/11 = 0.8 0.2048 (0.0 107) (0.0480)

Maize/Tobacco atpA 53 5 2/6 =0.3 0.1687 118 5 23/29= 0.8 0.6850 (0.06 1 5) (0.123 1)

atpH 101 7 8/8 = 1.0 0.1789 102 6 12/24=0.5 0.4784 (0.0466)

Wheat/Tobacco atpA 53 5 4/8 = 0.5 0.2703 107 7 18/25= 0.7 0.5893

atpH 101 9 8/11 =0.7 0.2170 106 5 14/30=0.5 0.6053

(0.0901)

(0.08 28) (0.1 101)

(0.052 1) (0.1074)

See the legend to Table 1 for details.

estimates of nucleotide divergence ( K ) are statistically homogeneous for the 5’- and 3’-flanking regions of atpA and atpH in maize and wheat. This indicates that these four regions are evolving at about the same rate in these two species. Second, the 5 ’ - and 3’-flanking regions of both genes each appear to be evolving at about the same rate in tobacco, though the former regions are evolving faster than the latter. Third, each of the four flanking regions is considerably more conserved in maize and wheat than tobacco.

Putative regulatory sequences involved in translation initiation, transcription initiation, and transcription termination are present in the flanking regions at atpA and atpH, but these sequences appear to show intergenic and interspecific variations:

Translation initiation: Sequences complementary to the 3‘ end of the plastid 16 S rRNA from maize (SCHWARZ and KOSSEL 1980) and tobacco (TOHDOH and SUGIURA 1982) are present immediately upstream from the translation initiation codons at atpA and atpH in all three species (double underlines in Figure 2). It has been suggested that these sequences may function in a manner similar to the “SHINE-DALGARNO” ribosome-binding sequences of Escherichia coli mRNAs (SHINE and DALGARNO 1974; BOGORAD et al . 1984). If this is the case, the data in Figure 2 indicate that there are qualitative and quantitative differences in the “SHINE-DALGARNO” sequences which are both gene-specific and species-specific (e.g., the “SHINE- DALGARNO” sequences are located further upstream from the translation initiation codon in both wheat genes). These differences may be a reflection of contrasting constraints on translation initiation since the efficiency of translation in Escherichia coli is correlated with the stability of the interaction between the mRNA and the rRNA, as well as with the spacing between the initiation codon and the “SHINE-DAL-

GARNO” sequence (STEITZ 1979). Consistent with these differences, the regions immediately flanking the “SHINE-DALGARNO” sequences in Figure 2 do not appear to be particularly conserved; these regions of Escherichia coli mRNAs appear to be important in determining translational efficiency because they, too, interact with the rRNA (STORMO et al . 1982; TANI- GUCHI and WEISSMANN 1978).

Transcription initiation: As illustrated by the S-1 nuclease mapping experiments in Figure 3, RNA termini are present in the 5’-flanking regions of both atpA and atpE from maize (arrows in Figure 2). Im- mediately upstream from both termini are sequences (underlined in Figure 2) which resemble putative “procaryotic-like,” “- 10” and “-35” promoter elements (ROSENBERG and COURT 1979) present upstream of the transcription start sites of other plastid genes (STEINMETZ et a l . 1983). Although S-1 nuclease mapping experiments have not been reported for these regions in wheat or tobacco, these sequences appear to be conserved upstream of aptA and atpH in all three species, with the exception of the 5’-flanking region of the tobacco atpH gene. The S-1 data do not rule out the possibility that the termini identified in the present experiments are RNA processing sites, rather than transcription initiation sites.

Transcription termination: Stable stem and loop structures, characteristic of rho-independent transcription termination in procaryotes (ROSENBERG and COURT 1979), can be formed in the 5’- and 3’-flanking regions of the tobacco atpA gene and in the 3’-flanking region of the tobacco atpH gene (DENO, SHINOZAKI and SUCIURA 1984; DENO and SUCIURA 1984), but similar structures cannot be formed in the flanking regions of either gene in maize or wheat. Since such a structural element appears to be lacking in the 3’- flanking region of atpA from maize (and wheat), it will


A

- . - - B

A A- A- A C ‘T

FIGURE 3.-S-1 nuclease mapping of RNA termini in the 5‘- flanking regions of A) the maize atpA gene and B) the maize olpH gene. The experiments were conducted as described in MATERIALS AND METHODS. As controls, RNA (‘-RNA”) or SI nuclease (‘-Sl”) were omitted from the reaction mixtures. The arrows indicate the protected bands.

be of interest to determine whether the convergently transcribed tRNAArg gene ( S . RODERMEL, P. ORLIN and L. BOGORAD, unpublished data; HOWE 1985) serves as a punctuation signal for transcription termination in this region of the plastid chromosome in either of these species.

DISCUSSION

According to the neutral theory of molecular evolution, genes (or parts of genes) evolve at rates com- mensurate with their functional importance (KIMURA 1983). Consistent with this theory, the present data show that the greatest number of nucleotide substitutions in the coding regions of uptA and uptH occur in the third codon position, largely as silent (synonymous) substitutions, and the smallest number in the second (nonsynonymous) codon position (Table 1). Rates of nucleotide substitution in mammalian pseudogenes are only somewhat greater than rates of synonymous substitution in the functional counterparts of these genes, suggvsting that the latter events are under little functional constraint (Lr, Luo and Wu 1985). Consequently, synonymous substitution rates provide a rough measure of the mutation rate in a given gene. This rate has been estimated as 1.1 X lo-” substitutions/site/year for rbcL, utpB and atpE in com-

parisons between maize and barley (ZURAWSKI and CLEGG 1984). and in the present studies estimates of this rate range from 0.43 to 1.9 X substitutions/ site/year, depending upon the gene, species comparison, and presumed divergence time (Table 2). These estimates a re significantly lower than similar estimates for genes from other sources-e.g., an average of 4.7 X 1 0-9 substitutions/site/year for mammalian nuclear genes (Lr, Luo and Wu 1985); 4.9 X substitutions/site/year for mammalian globin pseudogenes (LI, Luo and Wu 1985); and 100 X 1 0-” substitutions/ site/year for mammalian mitochondrial genes (e.g., BROWN et al. 1982; BROWN and SIMFSON 1982). T h e data are thus consistent with the notion derived primarily from comparative DNA hybridiiration and restriction fragment polymorphism studies that angiosperm plastid genomes are evolving conservatively in primary sequence with respect to other DNAs (reviewed in PALMER 1985a).

Although the third positions of synonymous codons a re presumed to be subject to few selective constraints, there is a marked tendency for these sites to be occu- pied by A or T in plastid genes (reviewed in WHITFELD and BOTTOMLEY 1983). In the present studies, 73% of the synonymous codons in atpA and 83% of the synonymous codons in atpH end in one of these two nucleotides (Table 4). Accompanying this AT-bias in third position codon usage is a transition mutation bias of about 2:l (Table 1). It has been observed previously that a twofold higher frequency of A and T in mammalian pseudogenes is likely the consequence of a nonrandom mutation bias favoring C to T and G to A transitions in the evolution of these sequences from their functional counterparts, suggesting that such a bias may be responsible for high equilibrium frequencies of A and T in selectively unconstrained regions of the mammalian nuclear genome (GOJOBORI, LI and GRAUR 1982; LI, Wu and Luo 1984). It is therefore tempting to speculate that a similar bias may be responsible (at least in part) for the fixation of high levels of A and T in relatively unconstrained regions of the plastid genome, including the third positions of synonymous codons (Table 1) and intergenic spacer regions (Table 3). Although a directionality of transition mutations cannot be in- ferred from the present data, analyses of plastid pseudogenes would clearly be of great benefit to test this hypothesis.

I t should be pointed out, however, that it is unlikely that a nonrandom mutation bias of this sort could alone account for the fixation of high levels of A and T in selectively unconstrained regions of the plastid genome, since even slight selective advantages appear to be capable of overriding such mutational pressures (Lr, Luo and Wu 1985). For example, examination of the patterns of codon usage in atpA and utpH from


TABLE 4

Codon usage in maize plastid genes

atpA atpH Genome

Ala GCU 26 GCC 9 GCA 12 GCG 2

Arg CGU 5 CGC 8 CGA 5 CGG 3 AGA 10 AGG 3

Asn AAU 12 AAC 1

Asp GAU 16 GAC 3

Cys UGU I UGC 0

Gln CAA 25 CAG 10

Glu GAA 31 GAG 10

Gly GGU 12 GGC 6 GGA 9 GGG 13

His CAU 2 CAC 2

Ile AUU 30 AUC 4 AUA 12

Leu UUA 16 UUG 8

I O 228 1 45 3 105 3 39

0 74 0 26 0 85 0 10 2 65 0 9

1 103 1 61

0 131 0 44

0 16 0 6

2 120 1 41

4 190 0 49

5 155 0 41 4 147 2 58

0 71 0 31

5 206 0 65 1 89

3 177 2 91

atbA atbH Genome

Leu CCU 12 3 CUC 6 0 CUA 5 3 CUG 4 1

Lys AAA 19 1 AAG 3 0

Met AUG 1 1 2

Phe UUU 7 3 uuc 7 0

Pro CCU 8 1 ccc 5 1 CCA 2 2 CCG 1 0

Ser UCU 8 1 UCC 12 1 UCA 8 0 UCG 2 0 AGU 4 1 AGC 1 0

Thr ACU 8 2 ACC 6 0 ACA 10 1 ACG 3 0

Trp UGG 0 0

Tyr UAU 13 1 UAC 3 0

Val GUU 6 4 GUC 3 0 GUA 17 2 GUG 7 1

Ter UAA 1 1 UAG 0 0 UGA 0 0

82 21 74 36

152 38

114

158 100

81 42 61 20

76 78 58 10 76 22

112 63 60 44

92

101 29

122 22

108 42

8 5 1

Codon usage in the maize atpA and atpH genes, expressed as the total number of codons for each amino acid. The codon usage in other maize plastid genes is also shown. These genes include: rbcL (MCINTOSH, POULSEN and BOCORAD 1980); atpB and atpE (KREB- BERS et al. 1982); P s l A l and pslA2 (FISH, KUCK and BOGORAD 1985); psbA and rps19 (I. LARRINUA, personal communication); psbC and psbD (E. T. KREBBERS, K. M. T . MUSKAVITCH, M. CAS- TROVEIJO, H. ROY, D. RUSSELL and L. BOGORAD, unpublished data); rpS4 (SUBRAMANIAN, STEINMETZ and BOGORAD 1983); psbG (STEIN- METZ et al. 1986); and the gene for cytochrome b-559 (J. LUKENS and L. BOGORAD, unpublished data).

maize (Table 4) reveals that not all preferred synonymous codons end in A or T, as predicted if the AT- bias at this position were a consequence of such a mutational bias-e.g., UCC, rather UCA or UCU, is the preferred codon for serine in @ A . Since these patterns of codon usage are similar to those observed in other maize plastid genes (Table 4), the data are in accord with the “genome hypothesis” of GRANTHAM et al. (198O)-viz., that genes in a genome use the

same coding strategy with respect to choices among synonymous codons-and indicate that evolutionary forces other than nonrandom mutation are at least partially responsible for the selection of synonymous codons in maize plastid genes. The selection of synonymous codons corresponding to the most abundant isoaccepting tRNAs for a given amino acid, and of those codons which most favorably stabilize codon- anticodon interactions appear to be two of the most important of these factors in bacterial and yeast sys- tems (e.g., IKEMURA and OZEKI 1983). The importance of these factors in determining the patterns of codon usage in plastid genes has yet to be explored. On the other hand, it should be noted that optimal levels of A and T in these, as well as other relatively unconstrained regions of the plastid genome, may be a reflection of other selective advantages associated with high frequencies of these nucleotides-e.g., energetic and/or deoxyribonucleotide triphosphate pool requirements by the plastid transcription and/or replication machinery.

A central finding of the present studies is that the patterns of nucleotide substitution differ among plastid coupling factor genes. This is perhaps best exem- plified by the differing modes of sequence evolution of atpE and atpH. Nucleotide divergence in atpE is characterized by much lower rates of nonsynonymous substitution in the monocot lineage (Table 2 ) , suggesting that the epsilon subunit of CFI is more selectively constrained in the monocots. The data ai-e thus in accord with previous analyses of overall nucleotide and amino acid homologies suggesting that atpE is more conserved in the monocots (PALMER 1985a). It is not known whether these constraints are associated with functional differences, but consistent with this notion, differences have been reported in the catalytic activities of isolated CFI from maize versus spinach (W. J. PATRIE and D. MILES, personal communication). In contrast, the sequence evolution of atpH is characterized by the virtual absence of nonsynonymous substitutions, but by a significantly lower rate of synonymous substitution in the monocot lineage. Unless this latter difference is a statistical aberration, these data suggest that the proteolipid subunit of CFo is under equal functional constraint in both the monocots and dicots, but that synonymous codon selection may be more constrained in the former. In accord with previous observations showing that highly expressed genes tend to use codons corresponding to the most abundant isoaccepting tRNA species, and weakly expressed genes those codons corresponding to more rare tRNAs (e.g., GRANTHAM et al. 1981; GROSJEAN and FIERS 1982; BENNETZEN and HALL 1982), such a constraint for atpH could arise from differences in tRNA availability in the monocot versus dicot lineages. This of course assumes that the stoi-

Molecular Evolution of atflA,H 137

chiometry of coupling factor subunits, which favors the proteolipid subunit by at least a factor of two to one (NELSON 198 l), is not primarily a consequence of post-translational events--i.e., that atpH is a highly expressed gene relative to the other coupling factor genes. Regardless of the precise nature of the constraint on synonymous codon selection in monocot atpH genes, it will be of great interest to determine whether a similar phenomenon underlies the sequence evolution of plastid genes encoding other highly “expressed” and/or conserved proteins, such as the 32- kd herbicide-binding protein of photosystem I1 (WHITFELD and BOTTOMLEY 1983).

The levels of nucleotide substitution in the 5’- and 3‘-transcribed flanking regions of atpA and atpH equal (in the maize/wheat comparisons) or vastly exceed (in the monocot/dicot comparisons) synonymous substitution levels in these genes, indicating that these regions are relatively unconstrained in their sequence evolution. Consistent with the high rates of nucleotide substitution in these regions, the data in this report indicate that there are few putative regulatory sequences in these regions that are conserved among all three species either in their spatial arrangement (e.g., translation initiation sequences) or sequence complexity (e.g., sequences flanking RNA termini; transcription termination sequences). This may not be surpris- ing, since the number (and likely location) of transcript ends in these regions appear to differ from species to species (FLUHR, FROMM and EDELMAN 1983; DENO, SHINOZAKI and SUGIURA 1984; BIRD et al. 1985; RODERMEL and BOCORAD 1985). The present data are thus not inconsistent with the notion that the flanking regions of atpA and atpH likely contain variable numbers of “species-specific” regulatory sequences (e.g., photoregulated promoter sequences in maize).

Unless “species-specific” regulatory elements are numerous in the flanking regions of atpA and atpH, the question arises why the sizes of these regions appear to be so highly conserved. As illustrated in Figure 2, the flanking regions of atpA and atpH are characterized by numerous small insertion (deletion) events which do not, in general, lead to a significant gain of nucleotides in one sequence us. another. The predominance of such events (in contrast to large insertion/deletion, transposition, and/or inversion events) has been noted by others in the noncoding regions of other plastid genes (e.g., ZURAWSKI, CLEGG and BROWN 1984; TAKAIWA and SUGIURA 1982), suggesting that, in terms of frequency, such events constitute the dominant mode of structural evolution in these regions of the plastid genome. The data are thus in accord with results from comparative DNA hybridization and restriction site studies showing that in terms of size, gene content, and gene arrangement,

angiosperm plastid DNAs are evolving conservatively in structure (reviewed in PALMER 1985b). As noted earlier, the data in this report are also consistent with the hypothesis that higher plant plastid genomes are evolving conservatively in primary sequence with respect to other genomes. This is indicated not only by the low rates of synonymous substitution in atpA, atpB, atpE and atpH, but also by the rates of nonsynonymous substitution in these genes (Table 2), which overall are at the low end of the range of similar rates for numerous mammalian nuclear genes, as well as several plant nuclear genes (LI, LUO and Wu 1985; S. ROD- ERMEL and L. BOGORAD, unpublished data). On the other hand, the present studies show that the transcribed flanking regions of atpA and atpH are not highly conserved in sequence complexity from species to species. This appears to be the case for the flanking regions of other, though not all, plastid genes (e.g., ZURAWSKI, CLEGG and BROWN 1984; S. RODERMEL and L. BOGORAD, unpublished data). The data thus suggest that the plastid genome is not a static evolutionary entity, and that the plastid does not respond passively to adaptive signals generated by differing, and more rapidly evolving nuclear environments. Rather, the data indicate that the plastid chromosome is an active participant in the evolutionary process, and that the rapid evolution of the flanking regions of plastid genes may be one means by which novel regulatory strategies can arise for the coordinate expression of genes in both compartments.

The authors would like to express their appreciation to LIZ ORR and PEGGY SCHULTZ for technical assistance; to ANDRE STEINMETZ and ENNO KREBBERS for guidance in DNA sequencing and S-1 nuclease analyses; and to DANIEL VOYTAS for careful reading of the manuscript. This work was supported in part by research grants from the National Science Foundation and the National Institute of General Medical Sciences. 1: was also supported in part by the Maria Moors Cabot Foundation of Harvard University.

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